Aspects of phosphoinositide and calcium signalling in a human neuroblastoma cell line
thesisposted on 15.12.2014, 10:34 by Amanda Kate. Martin
In the current study, the human neuroblastoma, SH-SY5Y, was employed to investigate aspects of receptor-mediated phosphoinositide (PI) and Ca2+ signalling. This cell line expresses many characteristics of neuronal cells, particularly following cellular differentiation. The effects of retinoic acid (RA)-induced differentiation on phosphoinositide and Ca2+ signalling were examined here. Some emphasis was placed on the regulation of the signalling pathway following cellular depolarisation or protein kinase C (PKC) activation, since differentiation of neuroblastoma cells has been associated with an increased density of voltage-operated Ca2+ channels and alterations of the expression of PKC isozymes.;In both RA-differentiated and undifferentiated SH-SY5Y cells, depolarisation augmented receptor-mediated [Ca2+]i elevation and PIC activation, providing evidence for signal integration between PIC-linked receptors and depolarising stimuli. However, differences between RA-differentiated and undifferentiated SH-SY5Y cells were minimal. Activation of PKC by phorbol ester reduced receptor-mediated PI hydrolysis in RA-differentiated and undifferentiated SH-SY5Y cells. Conversely, inhibition of PKC activity enhanced PIC activation in RA-treated cells only, suggesting PKC may play a more active role in regulation of the PI pathway in real mammalian neurones compared with neuroblastoma cells.;RA-differentiation of SH-SY5Y cells resulted in an up-regulation of muscarinic and bradykinin receptors. Stimulation of either receptor type with agonists, methacholine and bradykinin, revealed that agonist-mediated PIC activation was enhanced in the RA-treated cells. Comparisons of the concentration-effect curves for PIC activation and Ca2+ mobilisation from stores revealed that whilst there is a receptor for agonist-mediated PIC activation in RA-differentiated cells, concomitant with up-regulation for receptor density, there is no corresponding receptor reserve for agonist-evoked Ca2+ mobilisation. This was despite amplification of the response, as concentration-effect curves for agonist-stimulated Ca2+ mobilisation lay to the left of those for PIC activation. These data suggest a complex relationship between PIC activation and Ca2+ mobilisation in the SH-SY5Y cell line.